This proposal aims to understand the process and functions involved in cytomegalovirus DNA replication. Cytomegalovirus remains an important viral pathogen in immunocompromized individuals such as organ transplant recipients and ADDS patients and the only licenced anti-CMV antiviral, gancyclovir, is directed at the DNA replication machinery. The first aim in the next grant period, is to study a cisacting replication initiation sequence (oriLYT) located near the ICP8 gene and to localize the minimal sequence elements necessary for origin function in transient electroporation/infection assays. The oriLYT sequence from both the human and murine cytomegalovirus genomes will be mapped and studied. After functionally important region(s) are defined, viral and cellular proteins that interact with oriLYT sequence elements will be identified and cloned. We will use direct functional assays and mutagenesis to investigate the activity of the known CMV origin within the viral genome and to search for other replication origins. The second aim is to identify the set of trans-acting viral genes necessary to replicate oriLYT plasmids carrying the human or murine CMV DNA replication origins by a transient replication assay. Genomic DNA clones as well as existing murine and human CMV full-length cDNA libraries made in an SV40-based eukaryotic expression vector will be screened to isolate and study replication functions believed to encode the DNA polymerase, processivity factor, single stranded DNA binding protein, helicase-primase and origin binding protein based on similarity to HSV-1 gene products. The candidate cDNA expression clones encoding the necessary functions will be assayed by transfection of CMV-permissive human fibroblast and U373-MG cells as well as nonpermissive Vero cells and COS-7 cells, a cell line which will produce high levels of each of the products. All of the replication functions will also be expressed as recombinant proteins in baculovirus vectors for study of enzymatic function. The final aim will be to develop genetic manipulation strategies to construct defined mutants disrupting replication gene function. Viral mutants will be generated in each of the known or putative replication functions to determine their role during viral growth. Approaches that rely on cell lines that complement essential replication functions and approaches that make use novel methods to disrupt gene expression are proposed. These investigations will generate new information on an important pathogen as well as provide basic information on how these large enveloped DNA viruses replicate their genomes.